Guignot Florence, Baril Gerard, Dupont Francis, Cognie Yves, Folch Jose, Alabart Jose Luis, Poulin Naty, Beckers Jean-Francois, Bed'hom Bertrand, Babilliot Jean-Marc, Mermillod Pascal
UMR INRA-CNRS-Université de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France.
Mol Reprod Dev. 2009 Feb;76(2):183-90. doi: 10.1002/mrd.20940.
The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo.
本研究的目的是检测从经微刀片切割获得的绵羊胚胎活检组织中提取的DNA进行预扩增后基因型诊断的准确性，并评估活检胚胎在玻璃化/复温后移植到受体后的活力。确定了性别和PrP基因型。性别诊断是在PEP-PCR后通过PCR扩增ZFX/ZFY和SRY序列进行的，而PrP基因型测定是在对包括密码子136、154和171的特定靶标进行特异性预扩增后进行的。在发情后第7天收集胚胎。囊胚和扩张囊胚在收集后立即进行活检，而致密桑葚胚在体外培养24小时后进行活检。89个活检胚胎通过玻璃化冷冻。新鲜和玻璃化的完整胚胎作为对照。提取活检组织的DNA并进行预扩增。96.6%的活检组织性别诊断有效，95.8%的密码子确定了PrP基因分型。胚胎移植后，活检胚胎、玻璃化对照胚胎和新鲜胚胎的产羔率之间没有显著差异（分别为54.5%、60%和66.6%）。活检胚胎和整个玻璃化胚胎的胚胎存活率没有差异（P = 0.38）。出生时，96.7%的诊断性别和95.4%的预先确定密码子是正确的。羔羊的PrP谱与亲本基因型一致。PEP-PCR结合性别诊断和巢式PCR结合PrP基因型预先确定是在移植前对绵羊胚胎进行基因分型的非常准确的技术。这些原始结果允许在冷冻保存胚胎移植前规划对痒病抗性基因型和后代性别的选择。
